1. Field of the Invention
The present invention relates to methods of obtaining genetic competence in non-competent Bacillus cells.
2. Description of the Related Art
Genetic competence is a physiological state in which exogenous DNA can be internalized, leading to a transformation event (Berka et al., 2002, Mol. Microbiol. 43: 1331-45), but is distinct from artificial transformation involving electroporation, protoplasts, and heat shock or CaCl2 treatment. Natural competence has been observed in both Gram positive and Gram negative bacterial species (Dubnau, 1999, Annual Rev. Microbiol. 53: 217-44), and the process requires more than a dozen proteins whose expression is precisely choreographed to the needs of each organism.
Several hypotheses have been proposed regarding the purpose of natural competence, and they can be summarized as DNA for food, DNA for repair, and DNA for genetic diversity (Dubnau, 1999, supra). The DNA for food hypothesis is supported by observations that competence is a stationary phase phenomenon that occurs when cells are nutrient limited, and often a powerful nonspecific nuclease is co-expressed with transformation specific proteins. Evidence for the second hypothesis comes from the fact that genes encoding DNA repair enzymes are coordinately expressed with those encoding DNA transport proteins. Lastly, the DNA for genetic diversity hypothesis proposes that competence is a mechanism for exploring the fitness landscape via horizontal gene transfer. Observations that competence is regulated by a quorum-sensing mechanism and that it is a bistable condition (Avery, 2005, Trends Microbiol. 13: 459-462) support this hypothesis.
Public databases now contain a multitude of complete bacterial genomes, including several genomes from different strains of the same species. Recent analyses have shown, using pairwise whole genome alignments, that different strains of the same species may vary substantially in gene content. For example, genome comparisons of Escherichia coli strains CFT073, EDL933, and MG1655 revealed that only 39.2% of their combined set of proteins (gene products) are common to all three strains, highlighting the astonishing diversity among strains of the same species (Blattner et al., 1997, Science 277: 1453-74; Hayashi et al., 2006, Mol. Syst. Biol. doi:10.1038:msb4100049; Perna et al., 2001, Nature 409: 529-33; Welch et al., 2002, Proc. Natl. Acad. Sci. USA 99: 17020-17024). Furthermore, the genome sequence of E. coli strain CFT073 revealed 1,623 strain-specific genes (21.2%). From comparisons of this type, it is clearly seen that bacterial genomes are segmented into a common conserved backbone and strain-specific sequences. Typically the genome of a given strain within a species shows a mosaic structure in terms of the distribution of conserved “backbone” genes conserved among all strains and non-conserved genes that may have been acquired by horizontal transfer (Brzuszkiewicz et al., 2006, Proc. Natl. Acad. Sci. USA 103: 12879-12884; Welch et al., 2002, supra).
In terms of practical utility, transformation via natural competence is an extremely useful tool for constructing bacterial strains, e.g., Bacillus, that may contain altered alleles for chromosomal genes or plasmids assembled via recombinant DNA methods. Although transformation of certain species with plasmids and chromosomal DNA may be achieved via artificial means as noted above (e.g., electroporation, protoplasts, and heat shock or CaCl2 treatment), introduction of DNA by natural competence offers clear advantages of simplicity, convenience, speed, and efficiency.
In Bacillus subtilis, only 5-10% of the cells in a population differentiate to a competent state (termed the K-state) via a process that involves quorum-sensing, signal transduction, and a cascade of gene expression (Avery, 2005, supra). At least 50 genes are known to be involved directly in competence, and as many as 165 genes are regulated (directly or indirectly) by the central transcription factor ComK (Berka et al., 2002, supra). The competence cascade in Bacillus subtilis consists of two regulatory modules punctuated by a molecular switch (FIG. 1) that involves ComS binding to the adaptor molecule MecA, thereby interfering with degradation of the transcription factor ComK by the ClpC/ClpP protease (Turgay et al., 1998, EMBO J. 17: 6730-6738).
Much less is known about competence in the closely related species Bacillus licheniformis. Thorne and colleagues (Gwinn and Thorne, 1964, supra; Leonard et al., 1964, J. Bacteriol. 88: 220-225; Thorne and Stull, 1966, J. Bacteriol. 91: 1012-1020) published a series of papers in the 1960s that described transformation of three auxotrophic mutants derived from Bacillus licheniformis ATCC 9945A via natural competence. Natural competence was observed only in three specific auxotrophic mutants, 9945A-M28 (gly−), -M30 (uncharacterized auxotroph), and -M33 (pur−). Numerous other auxotrophs derived from the same parental strain (ATCC 9945A) did not give rise to transformants including those with requirements for thiamine, lysine, arginine, methionine, tryptophan, histidine, uracil, adenine, or hypoxanthine, and 13 other uncharacterized auxotrophic requirements (Gwinn and Thorne, 1964, supra). Furthermore, these investigators were unable to demonstrate transformation via natural competence in Bacillus licheniformis ATCC 10716 (Gwinn and Thorne, 1964, supra). As suggested by the early work of Thorne and colleagues, most Bacillus licheniformis isolates do not manifest natural competence, and in recent years genetic transformation of many Bacillus licheniformis isolates has been achieved only via electroporation (Tangney et al., 1994, Biotechnol. Techniques 8: 463-466), conjugation (Herzog-Velikonja et al., 1994, Plasmid 31: 201-206), or protoplasting (Pragai et al., 1994, Microbiol (Reading) 140: 305-310). The reasons for the apparent lack of a competent state in Bacillus licheniformis are unknown.
Ashikaga et al., 2000, Journal of Bacteriology 182: 2411-2415, describe the ability of Bacillus subtilis subsp. natto to develop genetic competence and the expression of the late competence genes required for incorporation of exogenous DNA. Liu et al., 1996, Journal of Bacteriology 178: 5144-5152, describe the elevation of competence gene transcription and transformation efficiency in wild-type Bacillus subtilis by multicopy expression of comS. Tortosa et al., 2000, Molecular Microbiology 35: 1110-1119, demonstrate that disruption of the ylbF gene leads to a decrease in expression of comK and that overexpression of comS suffices to bypass the competence phenotype of a ylbF mutation.
Since Bacillus licheniformis is a species of industrial importance, engineering strains that manifest natural competence is highly desirable for construction of new and improved production strains. The availability of a turn-key method for inducing competence in poorly transformable Bacillus licheniformis strains would improve the speed and efficiency with which chromosomal markers/alleles and expression vectors could be introduced. As described herein, the terms poorly transformable and non-competent are used interchangeably, and these terms mean that the number of transformants per microgram of DNA is less than twice the spontaneous mutation frequency when using the methods for competence-mediated transformation in Bacillus subtilis or Bacillus licheniformis as described previously (Anagnostopoulos and Spizizen, 1961, J. Bacteriol. 81: 741-746; Thorne and Stull, 1966, J. Bacteriol. 91: 1012-1020; Gwinn and Thorne, 1964, supra).
The present invention relates to methods of obtaining genetic competence in non-competent Bacillus cells.